It is now generally agreed that multiple opioid receptors exist in the mammalian central nervous system, but the roles they play in various opioid-mediated effects, and particularly in analgesia, are still unclear. Recent studies indicate that three major levels or areas of the CNS are critical in the control of pain: the dorsal horn of the spinal cord, the rostroventral medulla (RVM), and the periaqueductal gray (PAG). Thus identifying the opioid receptor types present in these areas is critical to our understanding of analgesia. As an approach to determining opioid receptor multiplicity, we recently analyzed the displacement of three ligands somewhat selective for different receptors: dihydromorphine (DHM) (mu); D-ala2-D-leu5- enkephalin (DADLE) (delta); and ethylketocyclazocine (EKG) (kappa). Using a computer model system, we found the best fit was with 4 distinct sites, two specific for DHM, one for DADLE, and one for EKG. We proposed to continue this work by testing the ability of B-endorphin and dynorphin, and their fragments, to displace these three tritiated ligands, as well as ligands that are highly selective for particular sites. The latter will include 3H- Tyr-D Ala-Gly-NMe-Phe-Gly-ol (3H-DAGO), which is mu specific; 3H-D-Pen2,5-Enkephalin (3H-DPEN), delta-specific; and 3H-U-50,488H, kappa-specific. We will apply this technique to each of three major CNS regions that have been implicated in opioid analgesia, with studies being conducted in both naive and morphine-tolerant animals. We will also determine displacement after treatment of tissue with alkylating agents, such as B-CNA or B-FNA, which inactivate binding sites in a selective manner. Since B-endorphin and dynorphin may also interact with sites not labelled by any of these ligands, we will also determine binding directly, using the tritiated peptides. Finally, we will use cross- linking reagents to label covalently the B-endorphin receptors. They can then be identified by polyacrylamide gel electrophoresis in SDS, and after further purification, tested for cross-reactivity to antibodies to the mu opiate receptor.